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Non-destructive assay systems for detection of β-glucuronidase activity in higher plants
Abstract
β-glucuronidase (GUS) can be qualitatively assayed in seedlings and fully grown plants without injury or irreversible damage
by short term incubations in X-gluc or by spraying 4-MUG.
... Histochemical analysis is used for the localization of gene activity in intact cells and tissues. For the histochemical assay, β -Glucuronidase uses an external substrate, 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) for histochemical localization resulting in a blue precipitate at the site of enzyme activity (Jefferson, 1987;Schmidt et al., 1992). ...
Cassava (Manihot esculentaCrantz) is a tropical root crop that serves as a staplefoodand a vital source of incometo small holder farmers in the tropics. Despite its contribution to food security, cassava production and utilization is faced by several challenges that include post-harvest physiological deterioration, insect and disease susceptibilities and accumulation of cyanogenic glycosides. Cassava crop improvement by conventional breeding has failed to address these constraints because of unsynchronized flowering, lack of resistance genes, high heterozygosity, allopolyploidy and poor seed set. Genetic transformation which begins with the establishment of embryogenic callus cultures can be used as one of the ways to complement these challenges faced by cassava breeders. This study investigated the effects of explant source (immature leaf lobes and meristematic stem segments), auxins (2, 4-dichlorophenoxyacetic acid (2, 4-D) and picloram),and photoperiod (0/24 and 16/8 light/ dark) oncallogenesis and embryogenesis in five African cassava genotypes (KME 1, 08/080, 08/354, 08/274 and TMS 60444). Callus formation and embryogenesis were successfully achieved in both explant sources. The leaf explants recorded significantly higher frequencies (p < 0.05) of somatic embryogenesiscompared to the stem explants in all the five genotypes. This study showed that the 0/24 light/dark photoperiod was superior to the 16/8light/ darkcycle for both callogenesis and embryogenesis. Althoughstatistically insignificant, 8mg/l 2, 4-D was the best concentration for theinduction of embryogenesis in 08/354, TMS 60444, 08/274 and 08/080 while 10mg/l gave the best results for genotype KME 1. Forpicloram, 10mg/l showedthe best results for embryogenesis across all genotypes. This study also determined the effects of varying formulations ofBAP (6-Benzylaminopurine), NAA (α-Naphthalene acetic acid) and GA3(Gibberellic acid)on somatic embryo maturation and plant recovery of the selected cassava genotypes Embryos in the cotyledonary stage were incubated in maturation medium supplemented with five different combinations of plant growth regulators: BAP, NAA, and GA3. Significant differences (p < 0.05) were recorded in shoot formation frequencieswith combination 2mg/l BAP, 0.01mg/l NAA, 1.5mg/l GA3and combination 1mg/l BAP, 0.02mg/l NAA, 1.5mg/l GA3giving the highest rates. Transformability was determined by carrying out a histological GUS(β-glucuronidase) assay on callustransformed using Agrobacteriumtumefaciensstrain EHA 101 harbouring plasmid pTF 102 with a GUSvisual marker gene and a bialaphos selectable marker gene. All the genotypes were found amenable to Agrobacteriummediated transformation with TMS 60444 and 08/274 recording the highest transformabilities of 73.33% and 68.33% respectively. A positive polymerase chain reaction (PCR) amplification targetingthe GUSgene confirmed the transfer of thetransgenes into cassava cells.The validated regeneration and transformation protocols reported here can be used for the integration of desired traits in African cassava genotypes.
... Histochemical analysis is used for the localization of gene activity in intact cells and tissues. For the histochemical assay, β -Glucuronidase uses an external substrate, 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) for histochemical localization resulting in a blue precipitate at the site of enzyme activity (Jefferson, 1987;Schmidt et al., 1992). ...
Cassava (Manihot esculentaCrantz) is a tropical root crop that serves as a staplefoodand a vital source of incometo small holder farmers in the tropics. Despite its contribution to food security, cassava production and utilization is faced by several challenges that include post-harvest physiological deterioration, insect and disease susceptibilities and accumulation of cyanogenic glycosides. Cassava crop improvement by conventional breeding has failed to address these constraints because of unsynchronized flowering, lack of resistance genes, high heterozygosity, allopolyploidy and poor seed set. Genetic transformation which begins with the establishment of embryogenic callus cultures can be used as one of the ways to complement these challenges faced by cassava breeders. This study investigated the effects of explant source (immature leaf lobes and meristematic stem segments), auxins (2, 4-dichlorophenoxyacetic acid (2, 4-D) and picloram),and photoperiod (0/24 and 16/8 light/ dark) oncallogenesis and embryogenesis in five African cassava genotypes (KME 1, 08/080, 08/354, 08/274 and TMS 60444). Callus formation and embryogenesis were successfully achieved in both explant sources. The leaf explants recorded significantly higher frequencies (p < 0.05) of somatic embryogenesiscompared to the stem explants in all the five genotypes. This study showed that the 0/24 light/dark photoperiod was superior to the 16/8light/ darkcycle for both callogenesis and embryogenesis. Althoughstatistically insignificant, 8mg/l 2, 4-D was the best concentration for theinduction of embryogenesis in 08/354, TMS 60444, 08/274 and 08/080 while 10mg/l gave the best results for genotype KME 1. Forpicloram, 10mg/l showedthe best results for embryogenesis across all genotypes. This study also determined the effects of varying formulations ofBAP (6-Benzylaminopurine), NAA (α-Naphthalene acetic acid) and GA3(Gibberellic acid)on somatic embryo maturation and plant recovery of the selected cassava genotypes Embryos in the cotyledonary stage were incubated in maturation medium supplemented with five different combinations of plant growth regulators: BAP, NAA, and GA3. Significant differences (p < 0.05) were recorded in shoot formation frequencieswith combination 2mg/l BAP, 0.01mg/l NAA, 1.5mg/l GA3and combination 1mg/l BAP, 0.02mg/l NAA, 1.5mg/l GA3giving the highest rates. Transformability was determined by carrying out a histological GUS(β-glucuronidase) assay on callustransformed using Agrobacteriumtumefaciensstrain EHA 101 harbouring plasmid pTF 102 with a GUSvisual marker gene and a bialaphos selectable marker gene. All the genotypes were found amenable to Agrobacteriummediated transformation with TMS 60444 and 08/274 recording the highest transformabilities of 73.33% and 68.33% respectively. A positive polymerase chain reaction (PCR) amplification targetingthe GUSgene confirmed the transfer of thetransgenes into cassava cells.The validated regeneration and transformation protocols reported here can be used for the integration of desired traits in African cassava genotypes.
... Histochemical analysis of the GUS reporter enzyme was performed as described by Martin et al. (1992), with minor modifications. For extensive expression description, 19 d after germination (dag) seedlings grown on Petri dishes and reproductive organs of adult plants growing in soil were stained. ...
Cytokinin and auxin are key regulators of plant growth and development. During the last decade transport mechanisms have turned out to be the key for the control of local and long‐distance hormone distributions. In contrast with auxin, cytokinin transport is poorly understood.
Here, we show that Arabidopsis thaliana AZG2, a member of the AZG purine transporter family, acts as cytokinin transporter involved in root system architecture determination. Even though purines are substrates for both AZG1 and AZG2, we found distinct transport mechanisms.
The expression of AZG2 is restricted to a small group of cells surrounding the lateral root (LR) primordia and induced by auxins. Compared to the wild‐type (WT), mutants carrying loss‐of‐function alleles of AZG2 have higher LR density, suggesting that AZG2 is part of a regulatory pathway in LR emergence. Moreover, azg2 is partially insensitive to exogenous cytokinin, which is consistent with the observation that the cytokinin reporter TCSnpro:GFP showed lower fluorescence signal in the roots of azg2 compared to the WT.
These results indicate a defective cytokinin signalling pathway in the region of LR primordia. The integration of AZG2 subcellular localization and cytokinin transport capacity data allowed us to propose a local cytokinin : auxin signalling model for the regulation of LR emergence.
... Наиболее значимые публикации по GUS даны в списке литературы [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. ...
The most widely used protocols for detecting major reporter genes, GUS and GFP (AFP), in plants are briefly reviewed. The most essential characteristics of these reporter genes and the corresponding proteins are presented. Possible areas of their application are considered. Such a review would be useful for gene engineers, molecular biologists, and other researchers, especially those who produce and study transgenic plants.
... Arabidopsis transformants were selected on sterile media containing 50 g mL 1 hygromycin.-Glucuronidase (GUS) staining was performed according toMartin et al. (1992). Tissues were cleared in 70% ethanol at 22C with shaking. ...
A new subfamily of sucrose transporters from Arabidopsis (AtSUT4), tomato (LeSUT4), and potato (StSUT4) was isolated, demonstrating only 47% similarity to the previously characterized SUT1. SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast. The Km for sucrose uptake by AtSUT4 of 11.6 ± 0.6 mM was ∼10-fold greater than for all other plant sucrose transporters characterized to date. An ortholog from potato had similar kinetic properties. Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves. In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading. In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements.
... GUS activity was localized histochemically by standard protocols (Jefferson, 1987;Martin et al., 1992). Handsectioned tissues or whole plant parts were fixed in a 0.35% (v/v) formaldehyde solution containing 10 mm Mes, pH 7.5, and 300 mm mannitol for 1 h at 20°C, rinsed three times in 50 mm sodium phosphate, pH 7.5, and subsequently incubated in 50 mm sodium phosphate, pH 7.5, 2 mm 5-bromo-4-chloro-3-indolyl--d-glucuronide cyclohexylammonium salt, and 20% (v/v) methanol for 6 to 12 h at 37°C. ...
Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1,tydc4, tydc6, tydc8, andtydc9 in one group and tydc2,tydc3, and tydc7 in the other. Thetydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the β-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern oftydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydcpromoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducibletydc regulation are conserved across plant species.
... Whole plant GUS staining (Figure 2(a)) was conducted following the non-destructive GUS assay method (Martin et al., 1992). ...
Nitrogen is a key mineral nutrient playing a crucial role in plant growth and development. Understanding the mechanisms of nitrate uptake from the soil and distribution through the plant in response to nitrogen starvation is an important step on the way to improve nitrogen uptake and utilization efficiency for better growth and productivity of plants, and to prevent negative effects of nitrogen fertilizers on the environment and human health. In this study, we show that Arabidopsis NITRATE TRANSPORTER 2.5 (NRT2.5) is a plasma membrane-localized high-affinity nitrate transporter playing an essential role in adult plants under severe nitrogen starvation. NRT2.5 expression is induced under nitrogen starvation and NRT2.5 becomes the most abundant transcript amongst the 7 NRT2 family members in shoots and roots of adult plants after long-term starvation. GUS reporter analyses showed that NRT2.5 is expressed in the epidermis and the cortex of roots at the root hair zone and in minor veins of mature leaves. Reduction of NRT2.5 expression resulted in a decrease in high-affinity nitrate uptake without impacting low-affinity uptake. In the background of the high-affinity nitrate transporter mutant nrt2.4, an nrt2.5 mutation reduced nitrate levels in the phloem of N-starved plants further than in the single nrt2.4 mutants. Growth analyses of multiple mutants between NRT2.1, NRT2.2, NRT2.4, and NRT2.5 revealed that NRT2.5 is required to support growth of nitrogen-starved adult plants by ensuring the efficient uptake of nitrate collectively with NRT2.1, NRT2.2 and NRT2.4 and by taking part in nitrate loading into the phloem during nitrate remobilisation.
... Seed was sterilized, germinated, and grown hydroponically in Magenta jars containing 60 mL of standard inorganic medium in sterile conditions, which was replaced weekly. After an additional 2 d, plants were stained for in vivo GUS activity by adding 2 mL of 50 mmolÁL –1 phosphate buffer (pH 7.5) containing 0.2 mmolÁL –1 X-gluc (5- bromo-4-chloro-3-indolyl-b-glucuronic acid) and incubated for 24–48 h according to the protocol of Martin et al. (1992). In vitro GUS activity was measured according to Breyne et al. (1993) with minor modifications. ...
Nitrogen (N) is the most important factor limiting crop productivity worldwide. The ability of plants to acquire N from applied fertilizers is one of the critical steps limiting the efficient use of nitrogen. To improve N use efficiency, genetically modified plants that overexpress alanine aminotransferase (AlaAT) were engineered by introducing a barley AlaAT cDNA driven by a canola root specific promoter (btg26). Compared with wild-type canola, transgenic plants had in-creased biomass and seed yield both in the laboratory and field under low N conditions, whereas no differences were ob-served under high N. The transgenics also had increased nitrate influx. These changes resulted in a 40% decrease in the amount of applied nitrogen fertilizer required under field conditions to achieve yields equivalent to wild-type plants. Résumé : L'azote est le plus important facteur limitant la productivité des cultures, au monde. La capacité des plantes à obtenir l'azote à partir des fertilisants appliqués, constitue une étape critique limitant l'efficacité de l'utilisation de l'azote. Afin d'améliorer l'efficacité de l'utilisation de l'azote, les auteurs ont modifié génétiquement des plantes qui surexpriment l'aminotransférase de l'alanine (AlaAT), en introduisant un cADN de l'AlaAT de l'orge guidé par un promoteur du canola spécifique aux racines (btg26). Comparativement au canola de type sauvage, les plantes transgéniques montrent un rende-ment en biomasse et en graines, au laboratoire aussi bien qu'aux champs, sous de faible apport en azote, alors qu'on ob-serve aucune différence en présence d'apports élevés en azote. Les plantes transgéniques augmentent également leur influx en nitrates. Ces changements conduisent à une diminution de 40 % l'application de fertilisant azoté nécessaire, pour obte-nir des rendements équivalents à ceux du type sauvage.
... Sudan and his colleagues demonstrated that GUS is ubiquitously present in plants [36]. Over the past three decades, numerous reports have illustrated that there was GUS background activity in different plant species, including some model plants, such as Arabidopsis thaliana, Oryza sativa, Nicotiana tabacum and Zea mays [36,38,54,55,56]. Therefore, efforts have been focused on the suppression of the background activity [57,58,59]. ...
A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter.
... After staining, tissues were cleared in 70% ethanol. Viable GUS staining was performed in tissue culture as described (Martin et al., 1992). For the analysis of GUS sectors in seedlings, seeds were germinated on Murashige and Skoog medium as described (Kode et al., 2006). ...
In eukaryotes, many genes were transferred to the nucleus from prokaryotic ancestors of the cytoplasmic organelles during endosymbiotic evolution. In plants, the transfer of genetic material from the plastid (chloroplast) and mitochondrion to the nucleus is a continuing process. The cellular location of a kanamycin resistance gene tailored for nuclear expression (35SneoSTLS2) was monitored in the progeny of reciprocal crosses of tobacco (Nicotiana tabacum) in which, at the start of the experiments, the reporter gene was confined either to the male or the female parental plastid genome. Among 146,000 progeny from crosses where the transplastomic parent was male, 13 transposition events were identified, whereas only one atypical transposition was identified in a screen of 273,000 transplastomic ovules. In a second experiment, a transplastomic beta-glucuronidase reporter gene, tailored to be expressed only in the nucleus, showed frequent stochastic expression that was confined to the cytoplasm in the somatic cells of several plant tissues. This gene was stably transferred in two out of 98,000 seedlings derived from a male transplastomic line crossed with a female wild type. These data demonstrate relocation of plastid DNA to the nucleus in both somatic and gametophytic tissue and reveal a large elevation of the frequency of transposition in the male germline. The results suggest a new explanation for the occurrence of uniparental inheritance in eukaryotes.
... Histochemical Locallzation and Fluorometric Quantitation of GUS Activlty GUS activity was localized histochemically by standard protocols (Jefferson, 1987;Martin et al., 1992). Typically, sectioned tissues or whole plant parts were incubated in 75 mM sodium phosphate, pH 7.5, 1 to 2 mM X-gluc in 10% dimethylformamide, and 0.5°/o Triton X-100 for 6 to 12 hr at 37%. ...
In legumes, the synthesis of infection- and elicitor-inducible antimicrobial phytoalexins occurs via the isoflavonoid branch of the phenylpropanoid pathway. To study transcriptional regulation of isoflavonoid pathway-specific genes, we have isolated the gene encoding isoflavone reductase (IFR), which is the enzyme that catalyzes the penultimate step in the synthesis of the phytoalexin medicarpin in alfalfa. Chimeric gene fusions were constructed between 765- and 436-bp promoter fragments of the IFR gene and the beta-glucuronidase reporter gene and transferred to alfalfa and tobacco by Agrobacterium-mediated transformation. Both promoter fragments conferred elicitor-mediated expression in cell suspension cultures derived from transgenic plants of both species and fungal infection-mediated expression in leaves of transgenic alfalfa. Developmental expression directed by both promoter fragments in transgenic alfalfa was observed only in the root meristem, cortex, and nodules, which is consistent with the accumulation of endogenous IFR transcripts. However, in transgenic tobacco, expression from the 765-bp promoter was observed in vegetative tissues (root meristem and cortex, inner vascular tissue of stems and petioles, leaf tips, and stem peripheries adjacent to petioles) and in reproductive tissues (stigma, placenta, base of the ovary, receptacle, seed, tapetal layer, and pollen grains), whereas the 436-bp promoter was expressed only in fruits, seed, and pollen. These data indicate that infection/elicitor inducibility of the IFR promoter in both species and developmental expression in alfalfa are determined by sequences downstream of position -436, whereas sequences between -436 and -765 confer a complex pattern of strong ectopic developmental expression in the heterologous species that lacks the isoflavonoid pathway.
... Physical evidence for ectopic recombination: To physically characterize the observed ectopic HR events, we searched for germinal recombination in F2 seedlings derived from the 3'AGUS X 5'AGUS:Ac crosses. Of 73,000 double hemizygote F2 seedlings, we failed to find germinal recombinant plants using a viable staining screen for GUS (MARTIN et al. 1992). Therefore, we used PCR to amplify recombinant molecules in somatic tissues. ...
The prominent repair mechanism of DNA double-strand breaks formed upon excision of the maize Ac transposable element is via nonhomologous end joining. In this work we have studied the role of homologous recombination as an additional repair pathway. To this end, we developed an assay whereby beta-Glucuronidase (GUS) activity is restored upon recombination between two homologous ectopic (nonallelic) sequences in transgenic tobacco plants. One of the recombination partners carried a deletion at the 5' end of GUS and an Ac or a Ds element inserted at the deletion site. The other partner carried an intact 5' end of the GUS open reading frame and had a deletion at the 3' end of the gene. Based on GUS reactivation data, we found that the excision of Ac induced recombination between ectopic sequences by at least two orders of magnitude. Recombination events, visualized by blue staining, were detected in seedlings, in pollen and in protoplasts. DNA fragments corresponding to recombination events were recovered exclusively in crosses with Ac-carrying plants, providing physical evidence for Ac-induced ectopic recombination. The occurrence of ectopic recombination following double-strand breaks is a potentially important factor in plant genome evolution.
... With this reporter gene system, temporal and spatial expression patterns could be characterised in detail. Although non-destructive GUS assays have been described and used successfully in plants [11], other, more recently developed systems exploiting luciferase (LUC) and green £uorescent protein (GFP) have many advantages [12,13]. In principle, these systems could be used to monitor expression during developmental processes in a single cell. ...
Transgenic Phytophthora palmivora strains that produce green fluorescent protein (GFP) or beta-glucuronidase (GUS) constitutively were obtained after stable DNA integration using a polyethylene-glycol and CaCl2-based transformation protocol. GFP and GUS production were monitored during several stages of the life cycle of P. palmivora to evaluate their use in molecular and physiological studies. 40% of the GFP transformants produced the GFP to a level detectable by a confocal laser scanning microscope, whereas 75% of the GUS transformants produced GUS. GFP could be visualised readily in swimming zoospores and other developmental stages of P. palmivora cells. For high magnification microscopic studies, GFP is better visualised and was superior to GUS. In contrast, for macroscopic examination, GUS was superior. Our findings indicate that both GFP and GUS can be used successfully as reporter genes in P. palmivora.
... Histochemical assays for b-glucuronidase activity were performed as described (Martin et al., 1992). Tissues were cut into 235 mm pieces, incubated in GUS staining solution containing 100 mM sodium phosphate (pH 7), 10 mM EDTA, 3 mM K 4 [Fe(CN) 6 ], 0.5 mM K 3 [Fe(CN) 6 ], 0.1% (v/v) Triton X-100, 2 mM 5-bromo-4-chloro-3indolyl-b-D-glucuronic acid (X-Gluc) for 3-24 h at 37 8C. ...
Amino acids are regarded as the nitrogen 'currency' of plants. Amino acids can be taken up from the soil directly or synthesized from inorganic nitrogen, and then circulated in the plant via phloem and xylem. AtAAP3, a member of the Amino Acid Permease (AAP) family, is mainly expressed in root tissue, suggesting a potential role in the uptake and distribution of amino acids. To determine the spatial expression pattern of AAP3, promoter-reporter gene fusions were introduced into Arabidopsis. Histochemical analysis of AAP3 promoter-GUS expressing plants revealed that AAP3 is preferentially expressed in root phloem. Expression was also detected in stamens, in cotyledons, and in major veins of some mature leaves. GFP-AAP3 fusions and epitope-tagged AAP3 were used to confirm the tissue specificity and to determine the subcellular localization of AtAAP3. When overexpressed in yeast or plant protoplasts, the functional GFP-AAP3 fusion was localized in subcellular organelle-like structures, nuclear membrane, and plasma membrane. Epitope-tagged AAP3 confirmed its localization to the plasma membrane and nuclear membrane of the phloem, consistent with the promoter-GUS study. In addition, epitope-tagged AAP3 protein was localized in endodermal cells in root tips. The intracellular localization suggests trafficking or cycling of the transporter, similar to many metabolite transporters in yeast or mammals, for example, yeast amino acid permease GAP1. Despite the specific expression pattern, knock-out mutants did not show altered phenotypes under various conditions including N-starvation. Microarray analyses revealed that the expression profile of genes involved in amino acid metabolism did not change drastically, indicating potential compensation by other amino acid transporters.
... Under these conditions, they reported that with few exceptions, the GUS activity was detected in certain part(s) of the fruit wall, seed coat, endosperm or the embryos of the tested plants. In addition, a number of publications on plant transformation in diVerent plant species report 'background activities' of GUS (Bekkaoui et al. 1988; Janssen and Gardner 1989; Lee et al. 1988; Martin et al. 1990; Raineri et al. 1990; Wenzler et al. 1989; Woznaik and Owens 1994 ). In most of these reports on GUS activity in plants, the observation was limited to recording the blue color after prolonged incubation in the histochemical assay buVer. ...
The enzyme beta-glucuronidase (GUS) is well characterized in animals and microbes. However, this enzyme is not well studied in plants and is widely assumed to be absent in them. In this study we document the ubiquitous presence of GUS in the model plants Arabidopsis thaliana, Oryza sativa, Nicotiana tabacum and Zea mays and record its expression pattern. The pH of the assay buffer was found to be critical with pH 4.0 being optimum for detection in all the species. GUS in plants appears to be associated with growth. In general, younger regions of the organs showed more GUS activity than the older and more mature tissues. In Brassica juncea roots stained for GUS, intense blue color could be seen in the trichoblast cells and the growing root hair cells as compared to the non-root hair forming epidermal cells or the fully elongated root hairs. Cotton fibers showed high GUS activity during the initial phase of elongation while the seed coat, from which the fibers formed, did not stain for GUS activity. The activity in the fibers disappeared after they were fully elongated. The level of GUS activity increased 2.58 folds in leaf tissues of N. tabacum when cultured in MS medium supplemented with 6-benzylaminopurine, while gibberellic acid enhanced GUS activity 2.9 folds in the inter-nodal regions of rice in 12-h treatment. In addition, elongation of stem, root and root hairs in tobacco seedlings was strongly inhibited by the specific inhibitor of GUS, saccharo-1-4-lactone in a reversible manner. Taken together, these evidences suggest a probable association of plant GUS in cell growth.
... Plants used for GUS expression studies were grown on sterile plastic mesh in Magenta jars containing 100 mL of liquid Murashige and Skoog (MS) medium (Sigma) for 1 week. The roots and leaves were stained for GUS expression according to the protocol of Martin et al. (1992). For longitudinal and cross-sections, stained roots and leaves were fixed, dehydrated, embedded in paraffin and cut into sections 5 μm thick using a rotary microtome. ...
Summary Nitrogen is quantitatively the most essential nutrient for plants and a major factor limiting crop productivity. One of the critical steps limiting the efficient use of nitrogen is the ability of plants to acquire it from applied fertilizer. Therefore, the development of crop plants that absorb and use nitrogen more efficiently has been a long-term goal of agricultural research. In an attempt to develop nitrogen-efficient plants, rice (Oryza sativa L.) was genetically engineered by introducing a barley AlaAT (alanine aminotransferase) cDNA driven by a rice tissue-specific promoter (OsAnt1). This modification increased the biomass and grain yield significantly in comparison with control plants when plants were well supplied with nitrogen. Compared with controls, transgenic rice plants also demonstrated significant changes in key metabolites and total nitrogen content, indicating increased nitrogen uptake efficiency. The development of crop plants that take up and assimilate nitrogen more efficiently would not only improve the use of nitrogen fertilizers, resulting in lower production costs, but would also have significant environmental benefits. These results are discussed in terms of their relevance to the development of strategies to engineer enhanced nitrogen use efficiency in crop plants.
En la naturaleza, las orquídeas interactúan con hongos micorrícicos y endófitos que les proporcionan nutrientes y/o estimulan la germinación y desarrollo inicial, sin embargo, algunos de sus mecanismos de acción aún se desconocen. La utilización de plantas modelo como Arabidopsis thaliana permite dilucidar estos mecanismos fisiológicos de las plantas, en condiciones controladas, ya que se cuenta con mutantes y líneas reporteras para todo tipo de respuestas. Se determinó la liberación de exudados de hongos endófitos y micorrícicos mediante cambios en el pH de medio MS suplementado con púrpura de bromocresol. Se analizó la respuesta de líneas reporteras de Arabidopsis (DR5::GUS, LOX2::GUS, PR1::GUS, CycB1::GUS, EXP7::GUS) y el ecotipo silvestre Columbia (Col-0), a la liberación de exudados volátiles y/o difusibles de dieciocho aislados fúngicos; además de la eficiencia de estos hongos como promotores en el desarrollo vegetal, evidenciado a partir de tinciones histoquímicas realizadas para revelar la acción de los genes reporteros. Las variables evaluadas fueron biomasa de follaje y raíz (peso fresco), longitud de raíz primaria y número de raíces laterales. Todas las cepas tuvieron liberación de exudados, siendo los hongos endófitos Preussia mínima (1) y Fusarium sp. quienes presentaron mayor liberación. La mayoría de las cepas estimularon el crecimiento de área foliar y raíz principal, formación de raíces laterales y, en algunos casos, raíces adventicias. En general, los hongos de estudio promovieron las mejores respuestas de la línea Col-0 a la presencia de compuestos volátiles, siendo la interacción con el hongo Trichocladium sp. 2 la que se reportó la mayor biomasa (200 mg). Algunas cepas como Preussia minima y Tulasnella sp. promovieron la división y expansión celular, asociado a la producción de reguladores del crecimiento vegetal como las auxinas. Otras como Xylaria sp. y Neofabraea sp. desencadenaron mecanismos de defensa con producción de ácido jasmónico y/o salicílico. Los hongos asociados a orquídeas liberan exudados que promueven el desarrollo de la planta a través de mecanismos que involucran la producción de factores de crecimiento, la división y elongación celular y/o sustancias que desencadenan mecanismos de defensa de acuerdo a las líneas reporteras de Arabidopsis thaliana analizadas.
Cotton fibers are seed trichomes that make cotton unique compared with other plants. At anthesis, IAA, a major auxin in plants, accumulates in the fiber cell to promote cell initiation. However, many important aspects of this process are not clear. Here, auxin distribution patterns indicated by auxin-dependent DR5::GUS (β-glucuronidase) expression in cotton ovules were studied during fiber cell differentiation and cell initiation [-2 to 2 DPA (days post-anthesis)]. The nucellus and fiber cell were two major sites where auxin accumulates. The accumulation in the nucellus started from -1 DPA, and that in fiber cells from 0 DPA. Immunolocalization analysis further suggests that the IAA accumulation in fiber initials began before flower opening. Furthermore, we demonstrate that accumulated IAA in fiber initials was mainly from efflux transport and not from in situ synthesis. Eleven auxin efflux carrier (GhPIN) genes were identified, and their expression during ovule and fiber development was investigated. Ovule-specific suppression of multiple GhPIN genes in transgenic cotton inhibited both fiber initiation and elongation. In 0 DPA ovules, GhPIN3a, unlike other GhPIN genes, showed additional localization of the transcript in the outer integument. Collectively, these results demonstrate the important role of GhPIN-mediated auxin transport in fiber-specific auxin accumulation for fiber initiation.
The principle of using reporter genes in studying molecular processes in a living cell means that in the natural gene, a synthetic modification is introduced (or the protein coding sequence is deleted and replaced by another gene) in order either to simplify the detection of the gene product or to distinguish it from similar or identical genes in the genome. The use of reporter genes requires a method of gene transfer — either transient or stable. Reporter gene technology can take very different shapes according to how and how much of the gene is tagged, and how the tag is detected or assayed. If one considers a schematic representation of an eukaryotic gene, with its cis-regulating regions, core promoter, transcript leader, translation initiation site, coding sequence, and 3′ control regions, a reporter tag can replace the gene to nearly any desired extent. Figure 1 shows that by replacing the gene to various extents by reporter sequences, gene fusions that can be used to analyze various levels of control of gene expression, as well as protein trafficking, can be generated.
Four new methods (titrimetric and spectrophotometric, two each) which are accurate, precise and rapid are described for the determination of promethazine theoclate in bulk form and in tablets. The titrimetric methods are based on the oxidation of the drug by metavanadate in acidic condition followed by determination of the unreacted oxidant by titration with iron(II) ammonium sulphate (Method A) or titration of the vanadium(IV) formed, with cerium(IV) sulphate (Method B). The spectrophotometric procedures are also based on the oxidation of the drug with metavanadate followed by the estimation of unreacted oxidant after complexing with chromotropic acid and measuring the absorbance at 420 nm (Method C) or estimation of the vanadium(IV) formed, by reacting it with ferriin and measuring the resulting ferroin at 510 nm (Method D). Method A and Method B are applicable over the ranges 1-20 and 5-12 mg, respectively. Calculations in both methods are based on the reaction stoichiometry of 1:2 (drug: metavanadate). In spectrophotometry, Beer's law is obeyed over the ranges 0.0-125.0 and 10.0-60.0 μg mL-1 for method C and method D, respectively and the corresponding molar absorptivity values are 2.64x10 3 and 5.33x103 L mol-1 cm-1, respectively. The methods were successfully applied to the determination of promethazine theoclate in avomine tablets, and the results obtained were in the range of 97.73-102.24% of declared content with a relative standard deviation of 1.51-2.22%.
Isoflavonoids are believed to play important roles in plant-microbe interactions. During infection of alfalfa (Medicago sativa) leaves with the fungal pathogen Phoma medicaginis, rapid increases in mRNA levels and enzyme activities of isoflavone reductase, phenylalanine ammonia-lyase, chalcone synthase and other defense genes are observed within 1 to 2 hours. The phytoalexin medicarpin and its antifungal metabolite sativan increase beginning at 4 and 8 hours, respectively, along with other isoflavonoids. In contrast, during colonization of alfalfa roots by the symbiotic mycorrhizal fungus Glomus versiforme, expression of the general phenylpropanoid and flavonoid genes phenylalanine ammonia-lyase and chalcone synthase increases while mRNA levels for the phytoalexin-specific isoflavone reductase decrease. The total isoflavonoid content of colonized roots increases with time and is higher than that of uninoculated roots, but the accumulation of the antifungal medicarpin is somehow suppressed.
An isoflavone reductase genomic clone has been isolated, promoter regions have been fused to the reporter gene β-glucuronidase, and the promoter-reporter fusions have been transformed into tobacco and alfalfa. Using histological staining, we have studied the developmental and stress-induced expression of this phytoalexin-specific gene in whole plants at a more detailed level than other methods allow. The isoflavone reductase promoter is functional in tobacco, a plant which does not synthesize isoflavonoids. Infection of transgenic alfalfa plants by Phoma causes an increase in β-glucuronidase staining, as does elicitation of transgenic alfalfa cell cultures, indicating that this promoter fusion is a good indicator of phytoalexin biosynthesis in alfalfa.
Three simple, selective, accurate and reproducible procedures are described for the assay of promethazine theoclate in bulk form and in tablets. One titrimetric and two spectrophotometric methods are based on the oxidation of the drug with potassium iodate, and determination of either excess iodate or iodine released in the reaction. In the titrimetric method (Method A) the drug reacted with a known excess of iodate in sulphuric acid medium followed by the iodometric determination of residual oxidant. The spectrophotometric method (Method B) is based on the oxidation of the drug in sulphuric acid medium in the presence of chloride ions by a large excess of iodate and the iodate being reduced to iodine. The ICl-2 generated in this reaction is used to iodinate 2′,7′-dichlorofluorescein dye, and the red colour of the iodinated dye is measured at 525 nm. The other spectrophotometric method (Method C) also involves the oxidation of the drug in acid medium by a large excess of iodate with the liberation of iodine and the latter being extracted into carbon tetrachloride and measured at 520 nm. The methods were successfully applied to the determination of promethazine theoclate in tablets and the results obtained were in agreement with the label claim.
Cyproheptadine hydrochloride (CPH), methdilazine hydrochloride (MDH) and promethazine theoclate (PMT) were determined in their pure state and in pharmaceutical formulations by a simple spectrophotometric method. The determination was based on the formation of a charge-transfer complex between chloranilic acid as a π-acceptor and the studied drugs as n-donors in an acetonitrile-chloroform mixture. The spectra, various experimental parameters, the stoichiometry and the stability of the complexes formed were investigated. The complexes formed were found to absorb at 520 nm. Beer's law is obeyed in the concentration ranges 25-125, 20-100 and 25-160 μg ml-1, for CPH, MDH and PMT respectively. The corresponding values of molar absorptivity and Sandell sensitivity are 1.48 × 103, 1.56 × 103 and 1.75 × 103 1 mo1-1 cm-1 and 217.39, 212.44 and 284.63 ng cm-2, respectively. The applicability of the method was demonstrated by the determination of the studied drugs in commercial tablets and syrup, and the results were statistically evaluated.
A number of transformation systems have been developed to insert foreign DNA into the appropriate plant genome (nuclear or plastid) (discussed in Chap. 3). However, only a small fraction of the treated cells become transgenic, while the majority of the cells remain untransformed using any of these methods. Thus, effective selection and screening strategies are needed to pick up the rare transgenic lines from a pool of nontransformed cells or plants. To date, more than 50 marker genes and a few molecular techniques have been developed to serve this essential purpose.
Significance
Proper timing of flowering is a key factor in reproductive success. Certain plant species require prolonged cold exposure during winter to flower in the spring; however, how plants measure the duration of cold exposure is not well understood. In this study, we describe the identification of a methyltransferase that is required for proper measurement of the duration of cold exposure.
The reactions of 2,10-disubstituted phenothiazine derivatives with halide and thiocyanate complexes of metal ions, pyrocatechol violet and with some oxidants are discussed in the article. The structure, mechanism of complexation reaction and analytical application of some phenothiazine complexes with metal ions are presented. The attention is paid to practical applications of oxidation reactions of phenothiazines in titrimetric, spectrophotometric, kinetic-catalytic and flow analysis of metal ions and phenothiazines.
Previously, assay conditions for the GUS enzyme have required lethal and/or destructive conditions which do not allow for
the continued observation of living plants. The replacement of sodium phosphate buffer with potassium phosphate buffer and
the removal of potassium ferrocyanide and EDTA resulted in an assay for the GUS enzyme that is both nondestructive and nonlethal
to tobacco plants and therefore allows observations of tobacco roots through time.
A mutant screen was developed to isolate Arabidopsis thaliana mutants affected in the regulation of the nitrate assimilation pathway. A fusion between the tobacco Nii1 gene (that encodes a foliar nitrite reductase involved in nitrate assimilation) and the Gus reporter gene was introduced into A. thaliana, and shown to be properly regulated by nitrate. Moreover, β-glucuronidase (GUS) activity in the transgenic plants was essentially detected in the cotyledons and leaves, showing that the organ-specific expression of the tobacco Nii1 gene was retained in Arabidopsis. M2 plantlets derived from mutagenized seeds homozygous for the Nii-Gus fusion were screened by histochemical staining of whole plates for GUS activity after growth on nitrate or glutamine. About 250 progenies were screened, leading to the isolation of plants showing an enhanced or reduced staining compared to the control non-mutagenized plants. Several mutants were analyzed for the transmission of the phenotype to the M3 generation, as well as for levels of GUS or nitrite reductase activities or mRNA levels. A major problem encountered during the screening was the high background of false positives that reproducibly showed altered GUS histochemical staining compared to control plants and did not, however, display any changes in GUS activity levels. One interesting family of mutants was isolated that overexpressed GUS activity and Nii mRNA in the absence of nitrate. These mutants turned out to be cnx mutants impaired in the molybdenum cofactor biosynthesis that is necessary for nitrate reductase activity. These results may indicate that active nitrate reductase is necessary for a correct regulation of nitrate assimilation genes by nitrate.
The firefly luciferase, assayedin vivo with a low-light video camera, acts as a non-invasive, real-time reporter of the temporal and spatial regulation of gene
expression in single plants. Furthermore, the sensitivity of the luciferase assay in extracts of transformed plant tissue
makes it a particularly useful marker in transient or stable transformation experiments.
Isoflavonoids are believed to play important roles in plant-microbe interactions. During infection of alfalfa (Medicago sativa) leaves with the fungal pathogen Phoma medicaginis, rapid increases in mRNA levels and enzyme activities of isoflavone reductase, phenylalanine ammonia-lyase, chalcone synthase and other defense genes are observed within 1 to 2 hours. The phytoalexin medicarpin and its antifungal metabolite sativan increase beginning at 4 and 8 hours, respectively, along with other isoflavonoids. In contrast, during colonization of alfalfa roots by the symbiotic mycorrhizal fungus Glomus versiforme, expression of the general phenylpropanoid and flavonoid genes phenylalanine ammonia-lyase and chalcone synthase increases while mRNA levels for the phytoalexin-specific isoflavone reductase decrease. The total isoflavonoid content of colonized roots increases with time and is higher than that of uninoculated roots, but the accumulation of the antifungal medicarpin is somehow suppressed.
An isoflavone reductase genomic clone has been isolated, promoter regions have been fused to the reporter gene β-glucuronidase, and the promoter-reporter fusions have been transformed into tobacco and alfalfa. Using histological staining, we have studied the developmental and stress-induced expression of this phytoalexin-specific gene in whole plants at a more detailed level than other methods allow. The isoflavone reductase promoter is functional in tobacco, a plant which does not synthesize isoflavonoids. Infection of transgenic alfalfa plants by Phoma causes an increase in β-glucuronidase staining, as does elicitation of transgenic alfalfa cell cultures, indicating that this promoter fusion is a good indicator of phytoalexin biosynthesis in alfalfa.
In recent years, considerable progress has been made in genetic engineering of various plant species, both agronomically important
crops as well as model plants. The bases of this progress were, in addition to efficient transformation methods, the design
of appropriate signals regulating transgene expression and the use of selection marker or reporter genes. In most cases, a
gene of interest is introduced into plants in association with a selectable marker gene (nptII, hpt, acc3, aadA, bar, pat). Recovery of a transgenic plant is, therefore, facilitated by selection of putative transformants on a medium containing
a selection agent, such as antibiotic (nptII, hpt, acc3, aadA), antimetabolite (dhfr), herbicide (bar, pat), etc. On the other hand, use of reporter genes (cat, lacZ, uidA, luc, gfp) allows not only to distinguish transformed and non-transformed plants, but first of all to study regulation of different
cellular processes. In particular, by employing vital markers (Luc, GFP) gene expression, protein localization and intracellular
protein traffic can be now observed in situ, without the need of destroying plant.
A biolistic particle delivery system was used to genetically transform pollen tubes of three species of white pine (Pinus aristata, P. griffithii and P. monticola). The introduced plasmid DNA contained the GUS coding sequence flanked by the 35S CaMV promoter and NOS terminator sequences.
Successful gene delivery was demonstrated by transient GUS expression as evaluated by standard histochemical assay. Distance
of target specimens significantly influenced transient GUS expression in all three species of white pine. A target distance
of 6 cm resulted in a significant number of transformed pollen tubes in P. aristata and P. griffithii, while distances of 6 and 9 cm resulted in a significant number of transformed pollen tubes in P. monticola. Generally, the number of pollen tubes expressing GUS activity was higher in P. aristata than in P. griffithii and P. monticola. The possibility of using GUS-transformed pollen tubes in conjunction with in vitro fertilization in conifers was examined.
Gene expression in pollen tubes was also examined under electron microscopy where the X-glu reaction product occurred as large
crystalline electron-dense precipitates in the cytoplasm.
A procedure for direct gene transfer into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants is presented. By modifying the microspore culture method and adopting
the firefly luciferase (Luc) gene as a non-destructive marker, we could obtain stably transformed androgenetic embryos from
bombarded microspores. Luc-positive embryos were easily isolated from the large non-transformed population using a high-sensitivity
bioluminescent image analyzer. PCR and Southern blot analyses confirmed that the introduced transgene was integrated stably
into the genome of the selected embryos. Diploidized plants obtained from the haploid embryos were self-pollinated, and all
of the offspring tested were Luc-positive, indicating rapid fixation of the transgene which is characteristic of doubled haploids.
Many reporter genes, such as gfp, gusA, and lacZ, are widely used for research into plants, animals, and microorganisms. Reporter genes, which offer high levels of sensitivity and convenience of detection, have been utilized in transgenic technology, promoter analysis, drug screening, and other areas. Directed molecular evolution is a powerful molecular tool for the creation of designer proteins for industrial and research applications, including studies of protein structure and function. Directed molecular evolution is based mainly on in vitro recombination methods, such as error-prone PCR and DNA shuffling. The strategies of directed evolution of enzyme biocatalysts have been the subject of several recent reviews. Here, we briefly summarize successes in the field of directed molecular evolution of reporter genes and discuss some of the applications.
Living organisms have been continuously evolving by assimilating new genetic material from the environment. However, this progress is very slow and often limited to transfer of genetic materials among closely related species. Recent developments in molecular biology and gene transfer techniques enable researchers to move genetic information among a variety of living organisms. Gene transfer techniques for higher plants can be divided into two major methods: direct DNA transfer and Agrobacterium-mediated tumor inducing (Ti)-plasmid-vector methods. Direct DNA transfer methods have been used for the transformation of a wide variety of species, especially those plant species that are recalcitrant to transformation with Ti plasmid vectors. However, the direct DNA transfer method requires more manipulation and transformation efficiency is generally much lower compared to the Ti-plasmid-vector system. Furthermore, it appears that stability of introduced genes is lower when DNA was directly transferred. Therefore, Ti vectors are commonly used for transformation of most dicot plant species that are, in general, more readily transformed using Agrobacterium-mediated DNA delivery.
Sucrose synthase, an important enzyme in carbohydrate metabolism, catalyzes the reversible conversion of sucrose and UDP to UDP-glucose and fructose in vitro. To investigate the in vivo function of sucrose synthase, both the gene (Asus1) and a corresponding cDNA from roots of Arabidopsis were isolated. The Asus1 gene has homologies of 67-72% to sucrose synthase genes from other species. Histochemical GUS analysis of Arabidopsis and tobacco plants transformed with a 1.5 kb Asus1 promoter fragment transcriptionally fused to the beta-glucuronidase reporter gene showed that the Asus1 gene is expressed in the phloem of leaves, and in roots. Induction is found under conditions of limited ATP supply and increased demand for translocation of carbohydrates such as anaerobic or cold treatment. During anaerobiosis the increase in RNA level leads to increased sucrose synthase activity in roots. The expression pattern and regulation of the gene suggest that sucrose synthase is involved in the supply of energy for phloem loading in source tissues, and in metabolization of sucrose in sink tissues after unloading.
In 1987, Richard Jefferson et al. (1) demonstrated the application of a new reporter gene system in transgenic plants. The reporter gene was the uidA gene of Escherichia coli that encodes the enzyme β-glucuronidase (GUS). Since then, the uidA gene (commonly referred to as the gus gene) has become one of the most widely used reporter genes in plant molecular biology. (For a detailed description of the
gus gene, see
Chapter 1.)
Sucrose is the main transported form of assimilates, but, significantly, it also regulates a variety of processes such as photosynthesis and carbon or nitrogen storage. The effects of high sucrose levels are mediated directly by modulation of gene expression. The regulation of storage protein accumulation, here patatin from potato tubers, was used as a model system to study sucrose mediated signal transduction. The transcriptional regulation of patatin genes in conserved in transgenic Arabidopsis, as shown by the analysis of expression of two classes of patatin promoters fused to uidA. Two distinctly different patterns of gene expression were observed. In roots, class I promoter expression is strongly dependent on the exogenous supply of sugars. 3-O-methylglucose induction indicates that the sensor is located upstream of hexokinase. In contrast, the class II promoter is constitutively active in root tips and hydatodes. The progeny of a homozygous class I line was mutagenized with ethyl methane sulphonate and screened for signal transduction mutants using a non-destructive screening system for GUS activity. Four mutants showing reduced sucrose responses (rsr) and two mutants with modified expression patterns (mep) regarding the root tip were identified. In backcross analyses, it was shown that rsr1-1 carries a recessive trans mutation whereas rsr4-1 seems to be a semi-dominant trans mutation in sugar-mediated gene regulation.
The expression of the in planta-induced gene ipiO of the potato late blight pathogen Phytophthora infestans was analyzed during various developmental stages of its life cycle. ipiO mRNA was detected in zoospores, cysts, germinating cysts, and young mycelia, but not in sporangia or in old mycelia grown in vitro. ipiO is not only expressed in stages prior to infection but also during colonization of potato and tomato leaves. In disease lesions, ipiO mRNA was detected in the water-soaked area and the healthy-looking plant tissue surrounding it. In contrast, ipiO mRNA was not found in necrotized tissue or in sporulating areas of a lesion. To determine more precisely the location and time of ipiO gene expression in planta, cytological assays were performed using a P. infestans transformant expressing a transcriptional fusion between the ipiO1 promoter and the beta-glucuronidase (GUS) reporter gene. GUS staining was found specifically in the subapical and vacuolated area of tips of invading hyphae. The histochemical GUS assays demonstrate that ipiO is expressed during biotrophic stages of the disease cycle.
A new subfamily of sucrose transporters from Arabidopsis (AtSUT4), tomato (LeSUT4), and potato (StSUT4) was isolated, demonstrating only 47% similarity to the previously characterized SUT1. SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast. The K(m) for sucrose uptake by AtSUT4 of 11.6 +/- 0.6 mM was approximately 10-fold greater than for all other plant sucrose transporters characterized to date. An ortholog from potato had similar kinetic properties. Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves. In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading. In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements.
Active oxygen species (AOS) generated in response to stimuli and during development can function as signalling molecules in eukaryotes, leading to specific downstream responses. In plants these include such diverse processes as coping with stress (for example pathogen attack, wounding and oxygen deprivation), abscisic-acid-induced guard-cell closure, and cellular development (for example root hair growth). Despite the importance of signalling via AOS in eukaryotes, little is known about the protein components operating downstream of AOS that mediate any of these processes. Here we show that expression of an Arabidopsis thaliana gene (OXI1) encoding a serine/threonine kinase is induced in response to a wide range of H2O2-generating stimuli. OXI1 kinase activity is itself also induced by H2O2 in vivo. OXI1 is required for full activation of the mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 after treatment with AOS or elicitor and is necessary for at least two very different AOS-mediated processes: basal resistance to Peronospora parasitica infection, and root hair growth. Thus, OXI1 is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses.
Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to overcome this obstacle a modified prokaryotic indicator gene was constructed. The expression of this indicator gene and its use in analysing early events in Agrobacterium-mediated plant transformation are described. A portable intron, derived from a plant intron, was introduced into the beta-glucuronidase (GUS) gene. In transgenic plants containing this chimaeric gene the intron is spliced efficiently, giving rise to GUS enzymatic activity. Mapping of the splice junction indicates the exact removal of the intron. No GUS activity is detected in agrobacteria containing this construct due to the lack of a eukaryotic splicing apparatus in prokaryotes. Early phases after transformation of Arabidopsis cotyledon explants were analysed using this GUS-intron chimaeric gene showing that as early as 36 h after Agrobacterium infection significant GUS activity is detected. In vivo GUS staining of transformed cells clearly shows that quickly proliferating calli expressing GUS activity are formed, mainly at the cut surface. Minor transformation events occur however throughout the whole cotyledon. These data indicate that Agrobacterium-mediated T-DNA transfer to plants is much more efficient than has been judged from experiments where selection is applied immediately. The intron-containing GUS gene can be used as an optimised marker gene in transient and stable transformation experiments.
A detailed analysis of the expression of a chimeric gene, consisting of the upstream region of the nuclear photosynthetic gene ST-LS1, encoding a component of the water-oxidizing complex of photosystem II, fused to the coding sequence of beta-glucuronidase (GUS) as a reporter, is described. The expression of this chimeric gene at the cellular level was detected by histochemical methods and shows that the expression of this gene is correlated with the presence of chloroplasts. Interestingly, the GUS activity was not only detected in typical photosynthetic tissues, e.g. leaves and stems, but also in green roots containing chloroplasts. In contrast no activity was detected in neighbouring white root tissue which was devoid of chloroplasts. One can therefore separate the relative importance of the (morphological) differentiation steps responsible for the formation of tissues normally involved in photosynthesis, from the importance of the developmental stage (characterized by the presence of chloroplasts), for the expression of this nuclear photosynthetic gene. Our data strongly suggest that the developmental stage of the plastids is the primary determinant for the activity of this nuclear photosynthetic gene, although they do not yet allow the exclusion of the reverse type of control, i.e. control of the differentiation of the plastid by the expression of certain nuclear genes. A chimeric gene, consisting of the promoter of the 35S cauliflower mosaic virus (CaMV) gene and the GUS coding sequence, was used as a control throughout the experiments, confirming that the observed differential ST-LS1-GUS gene expression reflects the particular transcriptional regulation impacted on this gene by its cis-acting regulatory sequences.
Patatin is one of the major soluble proteins in potato tubers and is encoded by a multigene family. Based on structural considerations two classes of patatin genes are distinguished. The 5'-upstream regulatory region of a class I gene contained within a 1.5 kb sequence is essential and sufficient to direct a high level of tuber-specific gene activity which was on average 100- to 1000-fold higher in tubers as compared to leaf, stem and roots in greenhouse grown transgenic potato plants when fused to the beta-glucuronidase reporter gene. Histochemical analysis revealed this activity to be present in parenchymatic tissue but not in the peripheral phellem cells of transgenic tubers. Furthermore the promoter fragment can be activated in leaves under conditions that simulate the need for the accumulation of starch in storage organs, i.e. high levels of sucrose. The expression is restricted to both mesophyll and epidermal cells in contrast to vascular tissue or hair cells.
A mutant of Arabidopsis thaliana (L.) Heynh. which lacks leaf starch was isolated by screening for plants which did not stain with iodine. The starchless phenotype, confirmed by quantitative enzymic analysis, is caused by a single recessive nuclear mutation which results in a deficiency of the chloroplast isozyme of phosphoglucomutase. When grown in a 12-h photoperiod, leaves of the wild-type accumulated substantial amounts of starch but lower levels of soluble sugars. Under these conditions, the mutant accumulated relatively high levels of soluble sugars. Rates of growth and net photosynthesis of the mutant and wild-type were indistinguishable when the plants were grown in constant illumination. However, in a short photoperiod, the growth of the mutant was severely impaired, the rate of photosynthesis was depressed relative to the wild-type, and the rate of dark respiration, which was high following the onset of darkness, exhibited an uncharacteristic decay throughout the dark period. The altered control of respiration by the mutant, which may be related to the relatively high levels of soluble carbohydrate that accumulate in the leaf and stem tissue, is believed to be partially responsible for the low growth rate of the mutant in short days. The depressed photosynthetic capacity of the mutant may also reflect a metabolic adaptation to the accumulation of high levels of soluble carbohydrate which mimics the effects of alterations in source/sink ratio. The activities of sucrose phosphate synthase and acid invertase are significantly higher in the mutant than in the wild-type whereas ADP-glucose pyrophosphorylase activity is lower. This suggests that the activities of these enzymes may be modulated in response to metabolite concentrations or flux through the pathways.
Patatin is one of the major soluble proteins in potato tubers and is encoded by a multigene family. Based on structural considerations two classes of patatin genes are distinguished. The 5′-upstream regulatory region of a class I gene contained within a 1.5 kb sequence is essential and sufficient to direct a high level of tuber-specific gene activity which was on average 100- to 1000-fold higher in tubers as compared to leaf, stem and roots in greenhouse grown transgenic potato plants when fused to the β-glucuronidase reporter gene. Histochemical analysis revealed this activity to be present in parenchymatic tissue but not in the peripheral phellem cells of transgenic tubers. Furthermore the promoter fragment can be activated in leaves under conditions that simulate the need for the accumulation of starch in storage organs, i.e. high levels of sucrose. The expression is restricted to both mesophyll and epidermal cells in contrast to vascular tissue or hair cells.
A highly efficient and fast Agrobacterium-mediated leaf disc transformation system for the Arabidopsis thaliana L. genotype C24 was developed. This protocol is also amenable to other ecotypes - as could be shown for Landsberg erecta and Wassllewskija. Besides the hygromycin selection also the G418 and kanamycin selection were established. Furthermore the described procedure is appliable not only to leaf explants but also to expanded cotyledons which proved to be an excellent alternative as explant source for transformation experiments.
β-glucuronidase (GUS) can be assayed in the spent media of plant tissues transformed with some GUS gene fusions (Jefferson,
1988). This approach is based on the presence of GUS in the media of transformed plant tissues expressing the gene and can
be used to monitor the progress of transformation without destruction of the tissue under study.
Progress in plant molecular biology has been dependent on efficient methods of introducing foreign DNA into plant cells. Gene transfer into plant cells can be achieved by either direct uptake of DNA or the natural process of gene transfer carried out by the soil bacterium Agrobacterium. Versatile gene-transfer vectors have been developed for use with Agrobacterium and more recently vectors based on the genomes of plant viruses have become available. Using this technology the expression of foreign DNA, the functional analysis of plant DNA sequences, the investigation of the mechanism of viral DNA replication and cell to cell spread, as well as the study of transposition, can be carried out. In addition, the versatility of the gene-transfer vectors is such that they may be used to isolate genes not amenable to isolation using conventional protocols. This review concentrates on these aspects of plant molecular biology and discusses the limitations of the experimental systems that are currently available.
The 5'-upstream region of the class I patatin gene B33 directs strong expression of the beta-glucuronidase (GUS) reporter gene in potato tubers and in leaves treated with sucrose. Cis-acting elements affecting specificity and level of expression were identified by deletion analysis in transgenic potato plants. A putative tuber-specific element is located downstream from position -195. Nuclear proteins present in leaf and tuber extracts bind specifically to a conserved AT rich motif within this region. A DNA fragment between -183 and -143, including the binding site is, however, not able to enhance the expression of a truncated 35S promoter from cauliflower mosaic virus. Independent positive elements contributing to a 100-fold increase relative to the basic tuber-specific element are located between -228 and -195; -736 and -509, -930 and -736 and -1512 and -951. Sucrose inducibility is controlled by sequences downstream of position -228, indicating that the tuber-specific and sucrose-inducible elements are in close proximity.
A new member of the patatin gene family belonging to the class II subfamily was isolated and characterized by DNA sequencing. In order to study the expression profile of this gene, the promoter was fused to the beta-glucuronidase gene and transferred to potato and tobacco. Histochemical analysis revealed high expression in a few defined cells in potato tubers and in a specific layer of both potato and tobacco root tips. In contrast to the developmentally and metabolically regulated class I patatin gene B33 this gene was not inducible by elevated levels of sucrose. Expression of this chimaeric gene was also found in callus and suspension cultures of potato.
Plants are unique in many aspects of development, cell biology, biochemistry, and physiological response. Plant cells do not migrate during morphogenesis, and totipotency of cells from many organs shows that plant development does not require maternal storage of positional information. Plant cell division is unlike that in other eukaryotes; plant cells are connected to their neighbors by unique structures (plasmodesmata) and live encased in a singular type of matrix (the cell wall). Plant biochemistry involves not only the metabolic processes familiar in animals but also several types of photosynthesis, as well as synthesis of an extraordinary range of plant-specific chemicals, many used in defense against herbivores and bacterial and fungal plant pathogens. In response to their environment, plants utilize unique pathways of light and gravity detection; in both environmental response and development they use growth substances, or hormones, quite unlike those of organisms in other kingdoms. Not only are these hormones chemically different from those in animals, but their use does not involve specific endocrine organs. Many of the most perplexing aspects of plant physiology, development, and biochemistry are now coming to be studied by the methods of classical and molecular genetics.
A chimeric gene consisting of 1.3 kb of the 5' regulatory region of a member of the potato proteinase inhibitor II gene family, the coding region of the bacterial beta-glucuronidase (GUS) gene and 260 bp of the proteinase inhibitor II 3'-untranslated region containing the poly(A) addition site was introduced into potato and tobacco by Agrobacterium tumefaciens mediated transformation. Analysis of transgenic plants demonstrates systemic, wound-inducible expression of this gene in stem and leaves of potato and tobacco. Constitutive expression was found in stolons and tubers of non-wounded potato plants. Histochemical experiments based on the enzymatic activity of the GUS protein indicate an association of the proteinase inhibitor II promoter activity with vascular tissue in wounded as well as in systemically induced non-wounded leaves, petioles, potato stems and in developing tubers. These data prove that one single member of the proteinase inhibitor II gene family contains cis-active elements, which are able to respond to both developmental and environmental signals. Furthermore they support the hypothesis of an inducing signal (previously called proteinase inhibitor inducing factor), which is released at the wound site and subsequently transported to non-wounded parts of the plant via the vascular system from where it is released to the surrounding tissue.